Degradation of several polycyclic aromatic hydrocarbons by laccase in reverse micelle system

Science of the Total Environment xxx (xxxx) xxx

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Degradation of several polycyclic aromatic hydrocarbons by laccase in reverse micelle system Pengfei Xu a,1, Hao Du a,1, Xin Peng a,⇑, Yu Tang a, Yaoyu Zhou b, Xiangyan Chen a, Jia Fei a, Yong Meng a, Lu Yuan a a National & Local United Engineering Laboratory for New Petrochemical Materials and Fine Utilization of Resources, Key Laboratory of Chemical Biology and Traditional Chinese Medicine Research (Ministry of Education of China), Key Laboratory of the Assembly and Application of Organic Functional Molecules of Hunan Province, College of Chemistry and Chemical Engineering, Hunan Normal University, Changsha 410081, China b College of Resources and Environment, Hunan Agricultural University, Changsha 410028, China

h i g h l i g h t s

g r a p h i c a l a b s t r a c t

 The degradation rate of PAHs in

laccase reverse micelles at different pH, temperature and aqueous ionic strength was investigated and the highest degradation rate reached 68.9%.  The secondary structure of laccase in reverse micelle system was studied.  A mechanism of benzo(b) fluoranthene degradation in laccase reverse micelles was proposed.

a r t i c l e

i n f o

Article history: Received 29 June 2019 Received in revised form 12 October 2019 Accepted 12 October 2019 Available online xxxx Editor: Daniel C.W. Tsang Keywords: Oily sludge PAHs Laccase Reverse micelles Degradation pathway

a b s t r a c t Remediation of polycyclic aromatic hydrocarbons (PAHs) in oily sludge has become the focus of attention. UV spectrophotometer analysis showed that four types of PAHs were found in sample, which including phenanthrene, anthracene, benzo(a)anthracene and benzo(b)fluoranthene. In order to degrade PAH effectively, the laccase reverse micelles system was proposed. The system protects laccase from being affected by organic phase. Reverse micelles were prepared by using isooctane to simulate oil. The optimum water content W0 was 10 by measuring the electrical conductivity of the system. Under this condition, the effects of pH, temperature and ionic strength on the degradation rate of PAHs were investigated. Also, compared with that of non-immobilized laccase, the ratio between the secondary structures of laccase under different conditions was studied. The results showed that the highest laccase activity was obtained at pH 4.2 and 30 °C with 60 mmol/L KCl. Meanwhile, the structure of a-helix accounts for the largest proportion, and the ratio of a-helix in the laccase secondary structure in the laccase-reverse micelle system was higher than that of the non-immobilized one under this condition. Finally, predicting the reactive site of the degradation of polycyclic aromatic hydrocarbons was simulated by ORCA (Version 4.2.0). The application in oily sludge was further conducted. This study provides an effective method and basis for the degradation of PAHs in oily sludge. Ó 2019 Elsevier B.V. All rights reserved.

⇑ Corresponding author. 1

E-mail address: [email protected] (X. Peng). Authors contributed equally.

https://doi.org/10.1016/j.scitotenv.2019.134970 0048-9697/Ó 2019 Elsevier B.V. All rights reserved.

Please cite this article as: P. Xu, H. Du, X. Peng et al., Degradation of several polycyclic aromatic hydrocarbons by laccase in reverse micelle system, Science of the Total Environment, https://doi.org/10.1016/j.scitotenv.2019.134970

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1. Introduction Oily sludge refers to sludge mixed with heavy oil, such as crude oil, various refined oil and residual oil. In the process of oil mining, refining, transportation and storage, a large amount of oily sludge can be produced. If the oily sludge is treated improperly, the air and groundwater will be polluted, and human health will be affected (Hu et al., 2013; Jasmine and Mukherji, 2015). The main components of the oil in the oily sludge are alkanes, polycyclic aromatic hydrocarbons (PAHs), asphaltenes, metal ions and resins. Among them, the adverse effects caused by PAHs in oily sludge are more serious (Huang et al., 2014). PAHs have high stability and are not easily degraded, and they can affect human beings through the food chain (Kim et al., 2013). PAHs are rapidly and widely distributed in the organism after absorption. With entering the lymph, PAHs circulate in the blood and are metabolized primarily in the liver. Some studies have demonstrated a series of health problems such as skin and lung bladder, and gastrointestinal cancers in workers exposed to PAHs mixtures (Abbas et al., 2018; Cetin et al., 2018; Kriipsalu et al., 2007). It is urgent to find a way to deal with PAHs. Environmental Protection Agency included 16 of PAHs in the list of priority pollutants (naphthalene, acenaphtalene, acenaphthene, fluorene, phenanthrene, anthracene, fluoranthene, pyrene, benzo(a)anthracene, chrysene, benzo(b)fluoranthene, benzo(k)fluoranthene, benzo(a)pyrene, indeno(1,2,3-cd)pyrene, dibenzo(a,h)anthracene, benzo(g,h,i)perylene) (Llobet et al., 2006). In recent years, researchers have paid more and more attention to the problems of PAHs pollution. Many methods have been developed, such as centrifugation, heat treatment, extraction, photocatalysis, oxidation, ultrasonic treatment and biodegradation(Cui et al., 2009; Hu et al., 2015; Kriipsalu et al. 2007; Lai et al., 2016; Leng et al., 2018; Lin et al., 2017; Rocha et al., 2010; Xu et al., 2009; Zheng et al., 2017). Among them, the principle of biodegradation is the use of biologically produced enzymes to degrade PAHs. This method is also the most environmentally friendly way. Laccase is a polyphenol oxidase containing four copper ions, widely found in bacteria, fungi and plants (Mayer and Staples 2002). Because of its excellent oxidation capability, laccase has been widely used in industry for degrading dyes, tetracycline, oils and phenols, etc. (Akkaya et al., 2016; Yang et al., 2017; Zhang et al., 2002). Apriceno et al. have proposed that laccase functioned well in the degradation of PAHs (Apriceno et al., 2017). The laccase produced by Trametes Versicolor has been used to degrade benzo(a) pyrene in soil, and the degradation rate of benzo(a)pyrene reached 15.6% (Zeng et al., 2018). The laccase produced by the fungus has been shown to degrade anthracene and benzo(a)pyrene in the soil(Wu et al., 2008). Furthermore, it is well known that the external environment is very easy to affect the activity of enzymes. So, in order to protect laccase activity, the free laccase can be fixed to improve the stability and better play the catalytic role (Chhaya and Gupte 2013). Enzyme immobilization on solid surfaces is one of the most common methods to improve enzyme activity and stability under harsh conditions over extended periods. Most traditional methods are immobilizing enzymes in magnetic mesoporous silica nanomaterials, electrospun fiber membranes and aldehyde supports materials (Addorisio et al., 2013; Dai et al., 2011; Song et al., 2012). Almost of these materials are used in the treatment of sewage, but rarely for oily sludge treatment. In practice, these materials should be directly exposed to the system. However, besides PAHs, there are metal ions, which will affect the activity of laccase in the oily sludge (Wang et al., 2018). The nano-scale agglomerates, which are called reverse micelles, with thermodynamic stability and transparent appearance formed spontaneously in organic solvents when the concentration of surfactant exceeds the critical micelle concentration (CMC) (Tonova

and Lazarova 2008). The surfactants in the reverse micelle system consist of two parts: hydrophilic group and hydrophobic group. The hydrophilic group aggregated into the polar nucleus, which could hold a part of the water to form a ‘‘pool”, and the polar molecules could be dissolved in this ‘‘pool”. The micro-water pools inside reverse micelles can solubilize hydrophilic biomolecules such as proteins, enzymes, DNA, and amino acids (Chen et al., 2017; Carvalho and Cabral, 2000; Ding et al., 2015). At the present, surrounding the reverse micelles in which the enzyme is participated, we have carried on a series of research (Peng et al., 2016). The effect of surfactant chain length on mixed reverse micellar extraction of cellulase was evaluated. The rhamnolipid (RL), a kind of biosurfactant, was used to form a novel reversed micellar system for extracting and purifying laccase from Coriolus versicolor crude extract (Peng et al., 2012). And compared with synthetic surfactants, the properties of the aqueous core as well as the microenvironment behavior and the laccase activity were investigated in reverse micelles formed by rhamnolipid (RL) (Cui et al., 2015). In addition, in this system, the parameters in degrading anthracene and pyrene were optimized for the purpose of improving degradation rates (Peng et al., 2015). While the purpose of this study is to evaluate the efficiency of laccase reverse micelles system in the degradation of PAHs observed in oily sludge. The fat-soluble substances which can affect the activity and stability of laccase can only maintain in the organic phase, which can prevent harmful substances from adverse effects on laccase and thus improve the activity of laccase. Most of the oil fractions of oily sludge are alkane compounds, so isooctane was used instead of oil in the first part of the experiment (Hu et al. 2013). The effects of pH, temperature and ionic concentration on the degradation efficiency were investigated. Then, the relationship between the secondary structure of laccase and the degradation rate was investigated. What’s more, we carried on some quantum chemistry calculations to predict the initial reaction site of the degradation of benzo(a)anthracene by laccase in reverse micelle and the degradation products were detected by GC–MC. In the end, the degradation efficiency of PAHs in oily sludge sample was determined directly by the system. 2. Materials and methods 2.1. Materials Phenanthrene, anthracene, benzo(a)anthracene, benzo(b)fluoranthene and hexadecyl trimethyl ammonium bromide (CTAB) were obtained from Aladdin. Laccase was obtained from Sigma. All the other chemicals used for the experiments and analyses were of AR grade. 2.2. Determination of PAHs in oily sludge A certain amount of oily sludge in dry beaker was froze and grinded into a powder. Then, regard the dichloromethane as extractant, the powder was extracted by Soxhlet extraction for 6 h. The obtained extractant was dried on a rotary evaporator and dissolved with a certain amount of cyclohexane. The extractant in cyclohexane was scanned by ultraviolet spectrophotometer at the range of 190–400 nm. 2.3. Determination of water content in reverse micellar system The reversed micelles which had different water content were prepared by injecting different amount of buffered stock solution into 0.5 g CTAB in /isooctane/ n-hexanol (10 ml:10 ml, v/v), while being vigorously shaken at given pH and concentration until a

Please cite this article as: P. Xu, H. Du, X. Peng et al., Degradation of several polycyclic aromatic hydrocarbons by laccase in reverse micelle system, Science of the Total Environment, https://doi.org/10.1016/j.scitotenv.2019.134970

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completely transparent solution was obtained. Then the conductivity of different moisture content was measured by conductivity meter, and the Origin software was used to draw the curve diagram of electrical conductivity which changed with the water content. And the best water content was judged from the graph. 2.4. Degradation of polycyclic aromatic hydrocarbons by laccase reverse micelle system The first part: A certain amount of phenanthrene, anthracene, benzo(a)anthracene and benzo(b)fluoranthene were dissolved by ultrasound in isooctane instead of oil in oily sludge. 0.5 g CTAB and 25 ml hexanol (the volume ratio of isooctane and hexanol was 1:1) were used as the oil phase of the reverse micelles, then the system was magnetically stirred for half an hour until the formation of reverse micelle. Then 0.1 mol/L citric acid and sodium citrate solution were used as buffer solution, and laccase buffer solution of 1 mg/ml was prepared as the water phase of reverse micelles. The mixture of oil and water was mixed to get laccasehexanol/isooctane reverse micelle solution. The water content in the system is expressed by W0 (the ratio of the total mole concentration of the buffer solution to the molar concentration of the surfactant). The properties of reverse micelle system were controlled by adjusting the pH, temperature and KCl concentration of the buffer solution. All reactions were completed in a 200 ml cone bottle, and the reaction lasted for 24 h. The samples under each condition were tested three times. The second part: The oily sludge was treated by ultrasonic extraction to obtain oil, and then 0.5 g CTAB and 25 ml n-hexanol were added and sonicated for half an hour until a reverse micelle was formed. Then added the laccase buffer solution under optimal conditions and stirred for half an hour. All reactions were completed in a 200 ml cone bottle, and the reaction lasted for 24 h. The samples under each condition were tested three times. 2.5. HPLC analysis The concentration of PAHs was analyzed by high performance liquid chromatography at 252 nm which was equipped withC18reverse-phase column (150  4.6 mm) and pump (P600). A mixture of methanol and deionized water (90:10) was used as mobile phase with 1 ml/min flow rate for PAHs analysis. The injection sample volume was 20 lL. The degradation rate of PAHs was calculated on the basis of comparison of peak area of sample chromatogram and peak area of standard chromatogram. 2.6. Characterization of secondary structure of laccase The secondary structure of laccase in the laccase reverse micelle system under different conditions was detected by circular dichroism. The different secondary structures of the enzyme protein have different characteristic peaks in the CD spectrum. The a-helix has characteristic peaks at 192, 208 and 222 nm, and the b-fold exhibits characteristic peaks at 216 nm and 185–200 nm. The b-turn showed a positive spectrum around 206 nm (Huang et al., 2007). The optical path of the circular dichroic sample cell was 0.1 cm; the sensitivity was 2 mdeg/cm; the scanning wavelength range was 190–250 nm; the scanning time was 0.5 s; the scanning speed was 3.3 nm/s; resolution was 0.1 nm. And the measurement was performed at room temperature. The CD spectrum was expressed by the average residue molar ellipticity in deg
cm2
dmol 1, and all circular dichroism data were averaged over 3 scans. After obtaining the CD spectrum, the content of the laccase secondary structure was calculated by using CDpro software. Firstly, the obtained data from CD spectrum need be input after opening the CRDATA. EXE. Then, there were several algorithms of which

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CONTINLL, SELCON3 and CDSSTR were the most frequently used and available online. We choose self-consistent method SELCON3 to calculate the secondary structure proportion of four batches of laccase. And the final results would be displayed in the PROSS.OUT. The reverse micelle system played a role in the immobilization of laccase, so the secondary structure of laccase in laccase buffer solution was tested according to the above method and compared with the secondary structure of laccase in reverse micelles. 2.7. Quantum chemical calculation With the development of quantum chemistry, density functional theory (DFT) has gradually formed. The basic idea is that the ground state physical properties of atoms, molecules and solids can be described by the particle density function. The DFT method can directly determine the exact ground state energy and electron density. Many researchers have explored the initial reaction through the FMO and FED determined by DFT (Barr et al., 2016; Chen et al., 2018; Li et al., 2015; Li et al., 2016; Qu et al., 2019; Qu et al., 2011a; Qu et al., 2011b; Shi et al., 2015; Wang et al., 2017; Zhuang et al., 2019). And the most common method is the B3LYP calculation method (Wei 2010). Since the B3LYP function can better reproduce the exact structure of the compound. In our experiments, the structural geometry was optimized by Gaussian 09 at the B3LYP/6-31G level (Ramos et al., 2007). The calculation of Hirshfeld charge (Zhou et al., 1953) and the fukui function (Yang and Parr 1985) was performed to predict the initial reaction site by using ORCA (Version 4.2.0) at the M06-2X/6-311g(d) level (Hehre and Lathan 1972; Zhao and Truhlar 2008). 2.8. GC–MS analysis Benzo(a)anthracene degradation metabolites were analyzed by GC–MS equipped with SH-Rxi-5Sil MS capillary column (30 m length, 0.25 mm internal diameter and 0.25 mm film thickness). The column temperature was programmed as follows: 60 °C for 2 min, an increase to 160 °C, at 15 °C/min, then again increased temperature to 300 °C at 25 °C/min and kept for 15 min at 300 °C. The injection sample volume was 1 lL in the splitless mode. The solvent delay time was set at 3 min. Injector and interface temperature was at 230 °C. 3. Results and discussions 3.1. Determination of the species of polycyclic aromatic hydrocarbons After UV detection, it can be seen from Fig. 1 that wave peaks were at wavelengths of 372, 350, 348, 346, 344, 313, 309, 299, 287, 282, 258, 251, 242 nm. By comparing those peaks with the standard PAHs absorption peaks (Spigno et al., 2007) (Table1), four types of PAHs were found in oliy sludge, including phenanthrene, anthracene, benzo(a)anthracene and benzo(b)fluoranthene. 3.2. The determination of the optimum water content The water content W0 in the system is the molar ratio of water to surfactant in the system. In reverse micelles, laccase exists in water phase, and there are two forms of water in the system: free water and bound water. When the free water is little, the conformation of laccase will be restricted, and its catalytic activity will be reduced. When the free water is much, the conformation of laccase will not be maintained. Therefore, it is necessary to find the best water content to maintain the activity of laccase (Senske et al., 2016; Tonova and Lazarova, 2008).

Please cite this article as: P. Xu, H. Du, X. Peng et al., Degradation of several polycyclic aromatic hydrocarbons by laccase in reverse micelle system, Science of the Total Environment, https://doi.org/10.1016/j.scitotenv.2019.134970

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Fig. 1. UV spectra of extracts dissolved in cyclohexane. Table 1 Maximum UV absorption wavelengths of 16 PAHs. PAH

UV absorption PAH wavelength/nm

UV absorption wavelength/nm

Naphthalene Acenaphtalene Acenaphtene Fluorene Phenanthrene Anthracene Fluoranthene Pyrene

219 228 225 210 251 251 232 238

287 267 258 240 295 296 210 250

Benzo(a)anthracene Chrysene Benzo(b)fluoranthene Benzo(k)fluoranthene Benzo(a)pyrene Indeno(1,2,3-cd)pyrene Dibenzo(a,h)anthracene Benzo(g,h,i)perylene

In reverse micelles, when applying an external electric field, the conductivity reflects the transport of the charged water cores whose ionization is driven by spontaneous thermal fluctuations. As with the increase of W0 from 2 to 10, the conductivity also increased. When W0 was 10, the conductivity was the highest, and the aqueous phase of the system had the best mobility, so the laccase activity will remain the best at this time. With the elevation of W0, the system became turbid from clarification gradually, and the viscosity of the system increased, leading to a decrease in conductivity (see Fig. 2). 3.3. Effect of pH on degradation rate and the secondary structure of laccase in the laccase-reverse micelle system Laccase activity plays an important role in the degradation of PAHs in laccase reverse micelles. The activity of laccase is easily lost in strong acid or strong alkali environment. When the pH value was 3.0–6.0, the degradation rate of PAHs was shown in Fig. 3. It can be seen that pH has a significant effect on the degradation effect. The degradation rate of pH increased gradually from 3.0 to 4.2 and decreased gradually at when pH above 4.2. When pH was 4.2, the activity of laccase was the highest, and the degradation rates of phenanthrene, anthracene, benzo(a)anthracene and benzo(b)fluoranthene reached the highest, which were 35.6%, 54.2%, 45.3% and 54.4%, respectively. The experimental results were similar to those obtained by Ma et al. (2018).

Fig. 2. Variation of electrical conductivity of reverse micelle system with water content.

The effect of pH on the secondary structure of laccase in the laccase-reverse micelle system was shown in Fig. 4(A). The results of CDpro calculation were shown in Table 2. According to the calculation results of CDpro, it can be seen that when the pH was in the range of 3.0–6.0, the proportion of a-helix increased first and then decreased, and the maximum proportion difference reached 30.4%. The b-sheet structure was first declined and then increased, and the largest proportion difference was 21.5%. However, the difference of b-turn angle and the random coil structure was not obvious in this range. At pH 4.2, the ratio of ahelix was the largest (49.1%), and the sum of the ratio of b-sheet and b-turn was 38.3%. The stability of the two level structure of proteins is directly related to the activity of laccase (Lópezcruz et al., 2006). The change of pH can affect the dissociation of the related groups on the active site of the enzyme, and change the original high regular structure in the molecule. At the same time, the secondary struc-

Please cite this article as: P. Xu, H. Du, X. Peng et al., Degradation of several polycyclic aromatic hydrocarbons by laccase in reverse micelle system, Science of the Total Environment, https://doi.org/10.1016/j.scitotenv.2019.134970

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Fig. 3. Effect of pH on the degradation of polycyclic aromatic hydrocarbons in laccase reverse micelles system; W0 = 10.

ture and the tertiary structure would also be affected by the change of pH, which leads to the change of the laccase activity. Li et al. (1999) have studied the proportion of the three structure under different pH conditions. The results show that the proportion of the three structure in the range of 4.0–6.0 is changed slightly. When the pH exceeds 6.0, the proportion of the random coil structure increases, so the activity of laccase is reduced. What’s more, at different sites of the enzyme protein polypeptide chain, there are amino acid residues. Thus, there is an electrostatic interaction between the side chain groups at different parts of the polypeptide chain. If it is a heterosexual charge, it helps to maintain the conformation of the protein; If it is repelled by the same charge, it is not conducive to the stability of the conformation of the protein, resulting in a loose conformation. For the protein, the lower the pH (below its isoelectric point) is, the more net positive charge is generated; when the pH is much higher than its isoelectric point, more net negative charge is generated. Because of the repulsion of the same charge and the breakage of the salt bond, the conformation of the protein molecule is loose (Xiao et al., 2004). In addition, irrespective of the pH value, the microenvironment will affect ionizing groups of the enzyme, and their ionization states will affect the interaction with the substrate seriously. The optimum pH of laccase depends largely on the protein residues, especially those closest to the active site. pH may also affect the microencapsulation of proteins. Usually the optimum pH is lower than isoelectric point (Carvalho and Cabral, 2000). In brief, the degradation rate of PAHs and the change of the ratio of secondary structure of laccase at different pH were consistent. It can be determined that the proportion of the secondary structure has a large influence on the activity of the laccase. 3.4. Effect of temperature on degradation rate and the secondary structure of laccase in the laccase-reverse micelle system Temperature is an important parameter affecting the degradation of PAHs. The change of the degradation rate of PAHs at temperatures ranging from 20 °C to 40 °C was shown in Fig. 5. From Fig. 5, it can be seen that the degradation rate increases gradually from 20 °C to 30 °C, and then decreases gradually after 30 °C. And the degradation effect was best at 30 °C. The degradation rate of phenanthrene, anthracene, benzo(a)anthracene, benzo(b)fluoranthene were 37.2%, 63.2%, 38.2% and 50.5% respectively. In general, enzyme-catalyzed reaction starts at a faster rate with increasing temperature up to a point, where the enzyme activity begins to decrease due to heat denaturation (Niu et al.,

Fig. 4. Circular dichroism of laccase at different pH (A), temperature (B) and KCl concentrations (C) in reverse micelle system.

2013). As the temperature increases, the molecules of the reactant can obtain energy so that some molecules of the lower original energy will become active molecules, and the percentage of activation molecules, the number of effective collisions and reaction rate will get increased. However, when the temperature is too high, the enzyme activity decrease, leading to the decline of degradation

Please cite this article as: P. Xu, H. Du, X. Peng et al., Degradation of several polycyclic aromatic hydrocarbons by laccase in reverse micelle system, Science of the Total Environment, https://doi.org/10.1016/j.scitotenv.2019.134970

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Table 2 Proportion of secondary structure of laccase at different pH in reverse micelle. pH

a-Helix

b-sheet

b-turn

Random coil

3.0 3.6 4.2 4.8 5.4 6.0

18.7% 36.8% 49.1% 43.5% 40.4% 19.8%

45.6% 39.8% 24.1% 24.7% 29.8% 30.2%

10.8% 10.5% 14.2% 16.0% 14.5% 20.5%

24.9% 12.9% 12.6% 15.8% 15.3% 29.5%

Fig. 5. Effect of temperature on the degradation of polycyclic aromatic hydrocarbons in laccase reverse micelles system; W0 = 10, pH = 4.2.

the proportion of other structures increases. There is no significant change in the b-turn and the random coil structure. When the temperature was 30 °C, the laccase activity was the highest, which was in consistent with the degradation rate of PAHs in the system. 3.5. Effect of aqueous ionic strength on degradation rate and the secondary structure of laccase in the laccase-reverse micelle system Another factor to be considered in the degradation of PAHs is the ionic strength. The ionic strength can affect the solubility of laccase in water nuclei and the activity of laccase. When the concentration of KCl was 20–100 mmol/L, the rate of degradation of PAHs was shown in Fig. 6. With comparison of Figs. 3, 5 and 6, it can be seen that the degradation rate of PAHs with different concentrations of KCl has been significantly varied. When the concentration of KCl was 60 mmol/L, the degradation effect was more obvious, and the degradation rate of phenanthrene, anthracene, benzo(a)anthracene and benzo(b)fluoranthene were 50.3%, 68.2%, 51.2% and 68.9%, respectively. And the effect of KCl concentration on the secondary structure of laccase in the laccase-reverse micelle system was shown in Fig. 4(C). The results of CDpro calculation were shown in Table 4. It can be seen from the Table 4 that in the KCl concentration range of 20–100 mmol/L the maximum proportion difference of a-helix was 27.6%, and the maximum proportion of b-fold structure was 11.9%. When the concentration of KCl was 60 mmol/L, the ratio of a-helix was up to 55.7%. Meanwhile, the sum of the ratio of b-sheet and b-turn was 32.9%, which is the lowest in the experiment. When the KCl concentration was 60 mmol/L, the ratio of a-helix was higher than that with the optimum pH (49.1%) and temperature (41.9%). Therefore, the degradation rate of PAHs and the change of the ratio of secondary structure of laccase at different KCl concentration were consistent.

efficiency (Niu et al. 2013; Zhu et al., 1998). Meanwhile, the relationship between temperature and the physical properties of the reverse micelles has been confirmed (Zulauf and Eicke 1979). For reverse micelles, high temperature will result in percolation, leading to changes in conductivity. Finally, a double continuous structure is formed, which will affect the activity of laccase. And the effect of temperatures on the secondary structure of laccase in the laccase-reverse micelle system was shown in Fig. 4 (B). The results of CDpro calculation were shown in Table 3. According to the calculation results, it can be seen that the ratio of the a-helix structure gradually increased when the temperature was from 20 °C to 30 °C. When the temperature exceeded 30 °C, the proportion of the a-helix structure gradually decreased, and the maximum difference was 29.5%. When the system temperature was 30 °C, the proportion of a-helix structure in the secondary structure of laccase was up to 41.9%, and the sum of the ratio of b-sheet and b-turn was the lowest (43.3%). When the temperature exceeds the optimum temperature, as the temperature increase, the hydrogen bond of the helical structure is destroyed, and the a-helix structure changes to the b-sheet, b-turn and random coil structure, so that the ratio of a-helix decreases gradually, and

Fig. 6. Effect of KCl concentration on the degradation of polycyclic aromatic hydrocarbons in laccase reverse micelles system; W0 = 10, pH = 4.2, T = 303 K.

Table 3 Proportion of secondary structure of laccase at different temperatures in reverse micelle.

Table 4 Proportion of secondary structure of laccase at different ion concentrations in reverse micelle.

Temperatures (°C)

a-Helix

b-sheet

b-turn

Random coil

C(KCl) (mmol/L)

a-Helix

b-sheet

b-turn

Random coil

20 25 30 35 40

12.4% 32.2% 41.9% 39.5% 37.3%

50.6% 32.1% 26.8% 28.9% 29.0%

22.4% 18.6% 16.5% 14.9% 16.7%

14.6% 17.1% 14.8% 16.7% 16.0%

20 40 60 80 100

28.1% 36.2% 55.7% 43.2% 39.9%

31.4% 29.2% 19.5% 22.6% 28.5%

20.2% 18.8% 13.4% 15.4% 15.8%

20.3% 15.8% 11.4% 18.8% 15.8%

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The interpretation for the following results may be due to that the presence of salts can reduce the inner surface potential, so the reactivity and stability of reverse micelle incorporating enzymes can be reduced (Biasutti et al., 2008). And there are two possible effects of salt (Zhu et al., 1998). One of them is that it can modify water properties, either favouring or limiting its dissolution by the micelles. The other is that the ionic strength of the buffer affects the spatial structure of the enzyme, thus affecting its catalytic activity. It has been mentioned in the foregoing that the hydrogen bond of a-helix plays a crucial role in the activity of laccase. Meanwhile, K+ can reduce the hydrogen bond change of ahelix and improve the stability of the secondary structure of laccase, thereby increasing the activity of laccase. In addition, based on the electrostatic shielding effect, increasing the ion concentration affects the interaction of the protein and the surfactant. This electrostatic shielding effect is also responsible for reducing the repulsion of the surfactant head, resulting in reduced water dissolution, and lead to the formation of smaller reverse micelles (AiresBarros and Cabral, 1991; Ichi-Ribault et al., 2017). When the ionic strength is low, the activity of enzymes dissolved in water phase can be maintained. With the increase of ionic strength and the decrease of the size of reverse micelle, the electrostatic attraction can reduce its thermal range of motion. In a short time, the distance between the enzyme molecule and the hydrophilic head of surfactant becomes shorter, which may lead to the structural change of laccase.

3.6. Degradation rate of polycyclic aromatic hydrocarbons and the secondary structure of laccase in non-reverse micelle systems As can be seen from the above results, the highest degradation rate of PAHs reached 68.9% when the laccase reverse micelles system pH = 4.2, the temperature was 30 °C, and the concentration of KCl was 60 mmol/L. A comparative experiment in which laccase was directly reacted with isooctane containing PAHs was carried out under optimal conditions. The degradation rates of phenanthrene, anthracene, benzo(a)anthracene and benzo(b)fluoranthene after 24 h were shown in Fig. 7. It can be seen from Fig. 7, that under the optimal conditions, the degradation rates of phenanthrene, anthracene, benzo(a)anthracene and benzo(b)fluoranthene were 19.6%, 19.8%, 20.5% and 21.1%, respectively. The degradation rate was much lower than that of the laccase reverse micelle system. Some scholars (Majcherczyk et al., 1998)also studied the degradation of PAHs by laccase. The degradation rates of phenanthrene, anthracene, benzo(a)anthracene and benzo(b)fluoranthene were 10%, 18%, 10% and 10%, respectively. And the degradation rate of other PAHs was also around 10%. The low degradation rate may be due to the effect of organic solvent on the activity of laccase. And the changes of secondary structure of laccase in nonreverse micelle system under different conditions were determined. Fig. 8 was circular dichroism chromatograms of laccases under different conditions in a non-reverse micelle system. The calculation results were shown in Tables 5–7. The results showed that the ratio of enzyme secondary structure in non-reverse micelle system was similar with that in the reverse micelle system. When pH was 4.2, the ratio of a-helix structure was 47.5%. However, the total ratio of b-fold and b-turn was 39.8%. When the temperature was 30 °C, the ratio of a-helix structure was up to 40.5%, and the proportion of b-sheet and bturn was 44.4%. When the concentration of KCl was 60 mmol/L, the ratio of a-helix structure was 54.4%, and the proportion of bsheet and b-turn was 33.8% together. Among them, the proportion of the random coil structure did not show distinct difference under different conditions.

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Fig. 7. Comparison of degradation rates of polycyclic aromatic hydrocarbons in reverse micelle system and non-reverse micelle system; W0 = 10, pH = 4.2, T = 303 K, C(KCl) = 60 mmol/L.

It can be seen from Tables 2–7 that under different conditions, the change of the random coil structure of the laccase was not obvious, and the main change was a-helix, b-sheet and b-turn, whether in reverse micelle system or non-reverse micelle system. When pH 4.2, temperature 30 °C and KCl concentration 60 mmol/L, the a-helix structure accounted for the largest proportion. However, the ratio between the secondary structures of laccase in the reverse micelle system differed from the secondary structure of laccase in the non-reverse micelle system. Due to the immobilization of the reverse micelle system, the ratio of a-helix of the laccase in the system was about 1–2% higher than that in the free state. From the secondary degradation rate and structure of laccase, it can be concluded that the ratio between the a-helix, b-sheet, bturn and random coil in the secondary structure was crucial for the laccase activity. The higher the proportion of a-helix, the higher the activity of laccase. The less the a-helix structure, the more the hydrogen bond. And other structures of the enzyme protein were destroyed, making the enzyme structure irreversible, so the laccase lost its catalytic activity. b-sheet and b-turn structure may cover the active site and active center of laccase to inhibit the binding of laccase to PAHs, leading to laccase inactivation and a decrease in the degradation rate of PAHs. Through the comparison of the degradation rate with PAHs in the reverse micelles, it can be seen that the reverse micelles system can be used to solve this problem well, which provides a better method for the degradation of PAHs. 3.7. Prediction of the initial reactions site by theoretical calculations The reaction of laccase with PAHs requires the participation of oxygen. According to the research, it is most likely that the PAHs are oxidized to form compounds with hydroxyl groups, and each carbon atom may undergo hydroxylation reaction. To predict the initial reaction site of the degradation of benzo(a)anthracene by laccase, the Hirshfeld charge and the fukui function were calculated using ORCA (Version 4.2.0) at the M06-2X/6-311g (d) level. As shown in Fig. 9A, the Hirshfeld charge in benzo(a)anthracene was mainly localized at C(6) ( 0.0453)and C(1) ( 0.04498). Therefore, it is possible for the C(6) and C(1) to combine new atom or atomic group with the formation of a radical intermediate. And in the process of degradation of benzo(a)anthracene, benzo(a)anthracene was confirmed that it was accepting electrons, the index

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P. Xu et al. / Science of the Total Environment xxx (xxxx) xxx Table 5 Proportion of secondary structure of laccase at different pH. pH

a-Helix

b-sheet

b-turn

Random coil

3.0 3.6 4.2 4.8 5.4 6.0

6.5% 35.0% 47.5% 45.2% 38.4% 11.9%

56.0% 40.2% 25.3% 25.4% 30.8% 31.1%

13.4% 12.1% 14.5% 15.0% 15.8% 19.5%

24.1% 12.7% 12.7% 14.4% 15.0% 37.5%

Table 6 Proportion of secondary structure of laccase at different temperatures. Temperatures (°C)

a-Helix

b-sheet

b-turn

Random coil

20 25 30 35 40

2.4% 30.2% 40.5% 38.2% 37.6%

58.6% 34.5% 29.4% 30.1% 30.6%

23.0% 18.3% 15.0% 15.6% 15.8%

16.0% 17.0% 15.1% 16.1% 16.0%

Table 7 Proportion of secondary structure of laccase under different KCl concentrations. C(KCl) (mmol/L)

a-helix

b-sheet

b-turn

Random coil

20 40 60 80 100

24.0% 35.2% 54.4% 42.5% 39.6%

33.1% 30.5% 20.6% 23.6% 28.8%

23.0% 18.9% 13.2% 14.9% 15.2%

19.9% 15.4% 11.8% 19.0% 16.4%

prone to take place at these positions. In addition, the contributions of each atoms at the HOMO and LUMO were displayed in Fig. 10. It shown that C(7) (13.18%) and C(8) (11.57%)had a major contributions at HOMO. And the occupied rates of C(7) and C(8) at LUMO were 11.49% and 11.13%, respectively. The calculation provided a similar result for prediction the initial reaction site of degradation of benzo(a)anthracene by laccase in reverse micelle, compared with the fukui function.

3.8. Analysis of benzo(a)anthracene degradation metabolites by GC– MS

Fig. 8. Circular dichroism of laccases in different pH (A), temperature (B) and KCl concentrations (C) in non-reverse micelle systems.

for nucleophilic attack. Generally, it will be represented by f+. Therefore, the f+was just calculated depending on the Hirshfeld charge. The f + value was mainly distributed at C(7) (0.073583), C (8) (0.069819), C(6) (0.054327) and C(1) (0.052051) (Fig. 9B), suggesting these C sites had higher reactivity. So the initial degradation of benzo(a)anthracene by laccase in reverse micelle is more

In this study, metabolites of benzo(a)anthracene degradation were conclusively analyzed by GC–MS. In the GC–MS analysis, the peaks of benzo(a)anthracene and metabolites of benzo(a)anthracene after degradation were shown in Fig. 11. The metabolites observed after degradation of benzo(a)anthracene (Fig. 11) include 4.343 (m/z 158, Butanoicanhydride), 8.915 (m/z 194, 1,2-Benzenedicarboxylicacid,1-ethylester), 10.473 (m/z 178, phenanthrene), 10.850 (m/z 222, Diethyl phthalate), and 12.766 (m/z 228, Benz(a)anthracene). On the basis of identified metabolites after GC–MS, the degradation pathway for benzo(a)anthracene was proposed in Fig. 12. Benzo(a)anthracene was oxidized by laccase to form intermediate 9,10-dihydroxy benzo(a) anthracene, then phenanthrene was formed by oxidation and ring breaking. Further the action of laccase, oxidation and ring cleavage reaction were occurred, and then diethyl phthalate was formed. This is similar to the result obtained by Tarafdar et al. (2018). The authors found the degradation metabolites of phenanthrene was phthalic acid, bis(2-pentyl) ester. Then diethyl phthalate converted into 1,2-benzenedicarboxylicacid,1-ethylester with the process of side chain hydrolysis. Butyric anhydride was formed by ring breaking and intermolecular dehydration.

Please cite this article as: P. Xu, H. Du, X. Peng et al., Degradation of several polycyclic aromatic hydrocarbons by laccase in reverse micelle system, Science of the Total Environment, https://doi.org/10.1016/j.scitotenv.2019.134970

P. Xu et al. / Science of the Total Environment xxx (xxxx) xxx

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Fig. 9. The Hirshfeld charge and the fukui function calculated by using ORCA (Version 4.2.0) at the M06-2X/6-311g (d) level. (A) The Hirshfeld charge, (B) The fukui function.

It was noteworthy that the phenanthrene was generated during the reaction, which may be the reason why the degradation rate of phenanthrene is low in the whole system. In many studies, quinones were first produced by laccase oxidation of PAHs. However, it did not appear in the actual detection, which may be caused by excessive metabolism of quinones (Acevedo et al., 2011). For intermediate products in degradation, they may be extracted from the system and used in industrial production. Butyric anhydride has many applications in practice, such as modifiers, photoactivatable prodrugs. Diethyl phthalate can be used as a carrier for perfume or cosmetics (Api, 2001; Soares et al., 2017; Zhang et al., 2017).

benzo(a)anthracene and benzo(b)fluoranthene were 13.9%, 14.8%, 9.6% and 10.2% respectively. The practical application of laccase degradation of PAHs has been reported, but most reports mention the degradation of PAHs in soil or water by laccase. However, due to the high cost of optimization of enzyme purification, fixation and the problem of medium in the soil, the degradation rate was not high. Some reports mentioned that the average degradation rate of PAHs was around 20% (Pan et al., 2011). Some studies have mentioned that redox mediators play an important role in the oxidation of PAHs. The addition of redox mediators such as ABTS and hydroxy benzotriazole to the system may increase the degradation rate in practical applications (Wu et al., 2008).

3.9. Practical application 4. Conclusion The oily sludge was extracted by ultrasonication to obtain an oil fraction, and an oil-reverse micelle system was prepared to degrade the PAHs existed in the sample. The experimental results were shown in Fig. 13. As can be seen from Fig. 13, under the optimal conditions, the degradation rates of phenanthrene, anthracene,

The reverse micelles were prepared by using isooctane to simulate the oil content in oily sludge in the first part, and the laccase reverse micelles system had good degradation effect on PAHs. This method reduced the influence of external factors on laccase activ-

Please cite this article as: P. Xu, H. Du, X. Peng et al., Degradation of several polycyclic aromatic hydrocarbons by laccase in reverse micelle system, Science of the Total Environment, https://doi.org/10.1016/j.scitotenv.2019.134970

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Fig. 10. The contributions of atoms at the HOMO and LUMO.

Fig. 11. The peak of benzo(a)anthracene degradation metabolites detected by GC–MS library search.

Please cite this article as: P. Xu, H. Du, X. Peng et al., Degradation of several polycyclic aromatic hydrocarbons by laccase in reverse micelle system, Science of the Total Environment, https://doi.org/10.1016/j.scitotenv.2019.134970

P. Xu et al. / Science of the Total Environment xxx (xxxx) xxx

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Fig. 12. The possible pathway of benz(a)anthracene degraded by laccase in reverse micelle system, based on the identified metabolites through GC–MS analysis.

tion site of the degradation of benzo(a)anthracene by laccase in reverse micelle. And we detected the degradation metabolites by GC–MS. Finally, reverse micelles were prepared from the oil in the oily sludge and the degradation of PAHs in the actual oily sludge was tested. This work has provided a good guidance for direct action on oily sludge in the future and the degradation products have little impact on the environment and are highly manageable. It may be possible to find a combination of physical, chemical and laccase reverse micelles to improve the degradation rate. Declaration of Competing Interest The authors declare that there are no conflicts of interest in the present experiment. Acknowledgements

Fig. 13. Degradation rate of polycyclic aromatic hydrocarbons in laccase-reverse micelle system in practical applications.

ity, played a better role in laccase catalysis and improved the utilization rate of laccase. The optimum conditions for the degradation of polycyclic aromatic hydrocarbons were found by optimizing various factors: the reaction time 24 h, the water content 10, the pH 4.2, the temperature 30 °C, and the KCl concentration 60 mmol/L. Under these conditions, the degradation rates of phenanthrene, anthracene, benzo(a)anthracene and benzo(b)fluoranthene reached 50.3%, 68.2%, 51.2% and 68.9% respectively. The proportion of the secondary structure of laccase has an important influence on its activity. The optimal conformation of laccase was clarified by comparing the proportions of different structures, and the superiority of reverse micelle system to laccase immobilization was also demonstrated. At the same time, we carried on some quantum chemistry calculations to predict the initial reac-

The authors acknowledge the financial supports from the National Natural Science Foundation of China (51608194), Hunan Provincial Natural Science Foundation of China (2019JJ50391), Scientific Research Fund of Hunan Provincial Education Department (18C0048 and 18C0071), Natural Science Foundation of Hunan (2018TP1017), and the Opening Fund of Key Laboratory of Chemical Biology and Traditional Chinese Medicine Research (Hunan Normal University), Ministry of Education. References Abbas, I., Badran, G., Verdin, A., Ledoux, F., Roumié, M., Courcot, D., Garçon, G., 2018. Polycyclic aromatic hydrocarbon derivatives in airborne particulate matter: sources, analysis and toxicity. Environ. Chem. Lett. 16, 439–475. Acevedo, F., Pizzul, L., Castillo, M.P., Cuevas, R., Diez, M.C., 2011. Degradation of polycyclic aromatic hydrocarbons by the Chilean white-rot fungus Anthracophyllum discolor. J. Hazard. Mater. 185, 212–219. Addorisio, V., Sannino, F., Mateo, C., Guisan, J.M., 2013. Oxidation of phenyl compounds using strongly stable immobilized-stabilized laccase from Trametes versicolor. Process Biochem. 48, 1174–1180.

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Please cite this article as: P. Xu, H. Du, X. Peng et al., Degradation of several polycyclic aromatic hydrocarbons by laccase in reverse micelle system, Science of the Total Environment, https://doi.org/10.1016/j.scitotenv.2019.134970